A major site of growth in the long bones of limbs is the epiphyseal plate. Here, chondrocytes progress from rapidly dividing cells to those undergoing hypertrophy and ultimate replacement. Hypertrophied cells initiate the synthesis of a collagen we have termed type X. When compared to known collagens, the type X molecule has unique properties. Its triple helix is about half the length of those of a "typical" interstitial collagen, has an uncommonly high thermal stability, and is readily cleaved at two sites by vertebrate collagenase. It has a COOH-terminal globular domain which is highly hydrophobic and contains a terminal sequence suggestive of a membrane-intercalated protein. It has the most restricted distribution of any known collagen, being synthesized largely, if not exclusively, by hypertrophic chondrocytes, and its initial deposition within the extracellular matrix seems to be regulated at the level of secretion. We now propose to determine some of the structural and functional roles of type X collagen, and examine the developmental mechanisms responsible for chondrocytes' initiating synthesis and secretion of this molecule. Structural studies will employ monoclonal antibodies for single- and double-label immunohistochemistry at both the light and electron microscopic levels. These will determine: (1) the form (supramolecular?) of type X within the extracellular matrix, (2) whether type X collagen is a component of matrix vesicles, and if so how it is organized within them, and (3) whether, during its synthesis and secretion, the molecule is segregated from other collagens and processed differently from them. We will also study the regulatory mechanisms involved in the cellular progression to type X synthesis by testing the hypotheses that certain components of cartilage matrix are inhibitory to this process, and that cell division is required. Lastly we will try to determine whether type X has a functional role in the assembly of matrix, possibly by "targeting" matrix vesicles and their contents to specific sites. As an additional/alternative hypothesis we will examine whether the molecule is involved in the degradation and/or removal of hypertrophic cartilage. These studies will involve matrixvesicles, mixed in vitro cultures of cartilage and blood-borne cells, chemotactic responses to the collagenase digestion products of type X, and the use of monoclonal antibodies as functional probes.